Although antibiotic therapy is now used for animal infections with some success, huge losses persist. The early hopes that antibiotics would allow complete control of the disease have not been realized. None of the antibiotics utilized thus far has been entirely satisfactory. Additionally, it has been found to be very desirable to replace antibiotic treatment with treatment by non-antibiotic chemo-therapeutic drug compounds, for the following reasons:
(1) Antibiotics effective in human medicine should not be utilized in veterinary medicine, in order not to build up strain resistance of bacteria appearing in human diseases; and PA1 (2) Antibiotics should be reserved for such diseases for which no chemo-therapeutic drug compound would be available, as it has been proved that bacterial strains build up resistance to an antibiotic after extended use of such antibiotic.
Despite these several published methods, it remains very important to find cost-effective methods utilizing non-antibiotic compounds which would substantially overcome the drawbacks of antibiotics used thus far and yet would be effective in treating and preventing infections in canine and feline animals.
Canine parvo virus still infects over one-half million young dogs. Hospitalization and intensive care are required. Mortality occurs in 15-20% of the cases. Severe neutropenia occurs and death is thought to frequently result from secondary infections and sepsis.
Feline Immunedeficiency Virus (FIV) is believed to infect 500,000-1,000,000 cats per year. This virus causes neutropenia in approximately 30% of the cats which renders them susceptible to infections. Feline Leukemia Virus (FeLV) also causes neutropenia in cats.
Granulocyte Colony Stimulating Factor
Granulocyte colony stimulating factor (G-CSF) is one of several glycoprotein growth factors known as colony stimulating factors (CSFs) because they support the proliferation of haemopoietic progenitor cells. G-CSF stimulates the proliferation of specific bone marrow precursor cells and their differentiation into granulocytes. It is distinguished from other CSFs by its ability to both stimulate neutrophilic granulocyte colony formation in semi-solid agar and to induce terminal differentiation of murine myelomonocytic leukemic cells in vitro. The cDNA cloning and expression of recombinant human G-CSF has been described, and it has been confirmed that the recombinant G-CSF exhibits most, if not all, of the biological properties of the native molecule (Souza, L. et al. Science 232, 61-65 (1986)). Sequence analysis of the cDNA and genomic DNA clones has allowed the deduction of the amino acid sequence and reveals that the protein is 204 amino acids long with a signal sequence of 30 amino acids. The mature protein is 174 amino acids long and possesses no potential N-linked glycosylation sites but several possible sites for O-linked glycosylation.
The cloning and expression of cDNA encoding human G-CSF has been described by two groups (Nagata, S. et. al., Nature 319, 415-418 (1986); Souza, L. M. et al., Science 232, 61-65 (1986)). The first report of a G-CSF cDNA clone suggested that the mature protein was 177 amino acids in length. The authors reported that they had also identified a cDNA clone for G-CSF that coded for a protein that lacked a stretch of three amino acids. This shorter form of G-CSF cDNA expresses the expected G-CSF activity. The second report describes a cDNA sequence identical to this short form and makes no mention of other variants. Since these authors confirmed that the short cDNA expresses G-CSF with the expected profile of biological activity, it is probable that this is the important form of G-CSF and that the longer form is either a minor splicing variant or the result of a cloning artifact.
Matsumoto et al., in Infection and Immunity, Vol. 55, No. 11, p. 2715 (1987) discuss the protective effect of human G-CSF on microbial infection in neutropenic mice.
The following patent publications relate to G-CSF: WO-A-8703689, assigned to Kirin/Amgen describes hybridomas producing monoclonal antibodies specific for G-CSF and their use in the purification of G-CSF; WO-A-8702060, assigned to Biogen, discloses human G-CSF like polypeptides and methods of producing them; U.S. Pat. No. 4,810,643 assigned to Amgen, discloses human G-CSF like polypeptides, sequences encoding them and methods of their production; and WO-A-8604605 and WO-A-8604506, both asigned to Chugai Seiyaku Kabushiki Kaisha, disclose a gene encoding human G-CSF and infection inhibitors containing human G-CSF.
The use of recombinant G-CSF with the same amino acid sequence as human G-CSF, in dogs with cyclic neutropenia has been associated with the development of neutralizing antibodies to the heterologous G-CSF protein during a thirty day period of administration (see Lothtop et al., Blood 72, 5624-37 (1988). Subsequent treatment of these same dogs with recombinant human GM-CSF failed to cause a significant leukocytosis or eliminate cycles of neutropenia. A significant variation in structure may explain the development of neutralizing antibodies when the human sequence products are given to dogs. The development of neutralizing antibodies in dogs given the human sequence products may limit them to single or short term use.
It is an object of the subject invention to provide an improved method of treating and preventing infections in canine or feline animals.
It is a further object of the subject invention to provide a method of treating infections in canine or feline animals without build up of strain resistance of bacteria.
A still further object of the invention is to provide a purified and isolated polypeptide having part or all of the primary structural conformation and the biological properties of naturally occurring canine G-CSF, and DNA sequences encoding such G-CSF.
Other objects, features and characteristics of the present invention will become apparent upon consideration of the following description and the appended claims.